Proceedings of The 2nd Annual International Conference Syiah Kuala University 2012

& The 8th IMT-GT Uninet Biosciences Conference

Banda Aceh, 22-24 November 2012

Antibacterial activity of red betel (Piper crocatum) leaf

methanolic extracts aginst methicillin resistant
Staphylococcus aureus
1
Tristia Rinanda, 2Zulfitri, 3Desi Maharani Alga
1
Microbiology Department, Faculty of Medicine, Syiah Kuala University 2Biology Department, Faculty of
Medicine, Syiah Kuala University, 3Medical student of Faculty of Medicine, , Syiah Kuala University.
Corresponding author: tristia_rinanda@yahoo.com

Abstract. Red betel (Piper crocatum) is one of traditional medicine that has antibacterial activity. This research was
conducted to determine the antibacterial activity of the methanol extract of red betle leaves against growth of Methicillin
Resistant Staphylococcus aureus (MRSA). This research used Completely Randomized Design (CRD) method with 5
treatment groups consist of red betle leaves extract with concentration 150 mg/mL, 300 mg/mL, 450 mg/mL, 600
mg/mL, and negative control (sterile water) with 4 times repetition. The methanol extract of red betle leaves was
obtained by maceration technique using methanol 96% as solvent. Antibacterial activity test conducted with Kirby Bauer
disc diffusion method. Data of this research were analyzed by ANOVA followed by Duncan Test. The results showed that
red betel leaf extracts in concentration 150 mg/mL, 300 mg/mL, 450 mg/mL, 600 mg/mL provide average inhibition
zone respectively 9,0 mm, 11,2 mm, 13,6 mm and 15,7 mm. The result showed that red betel leaf extracts in tested
concentration showed antibacterial activity against MRSA. The higher concentration of extract, the greater inhibition area
was formed.
Keywords: Red betel methanolic extract, MRSA, antibacterial activity

Introduction
MRSA bacterium was first found in the United States in 1968. It is included in emerging
infectious pathogen, and can spread through contacts between the infected medical workers
and patients in hospitals. In the last few decades, the incidents of MRSA infection have kept
increasing in many parts of the world (Arnita, 2007). A research conducted in 9 countries in
1974 showed that 2% of the overall S. aureus infection cases were MRSA. This figure went up
to 22% in 1995 and 85% in 2008, and has kept rocketing eversince (Sheen, 2010). A research
conducted by Maulana (2011) proved that S. aureus can be isolated from the sputum of
chronic coughing patients in RSUDZA Banda Aceh (9 isolates out of 100 speciments (9%).
From those 9 isolates, 5 isolates (5%) were identified as MRSA. MRSA mortality rate reached
50% in the period of 1999-2005 in all American hospitals which far exceeded the increase of
mortality rate of Methicillin-Susceptible Staphylococcus aureus (MSSA) which was only 18% in
the same period (Klein et al., 2007).
An increased bacterial resistance to the existing antibiotics should be balanced with the
discovery of new medication. An alternative product which is more potent and low-cost, with
fewer side effects and continuous large number availability needs to be discovered to overcome
antibiotic resistance. One way of doing this is by using a antibacterial active substance
contained in the medicinal plants. One of medicinal plants commonly used by people is betel
plant. Betel plant has been known as an antiseptic since 600 BC. The types of betel generally
used as medicine in Indonesia are green betel (Piper betel) and black betel. However, there is
another type gaining popularity i.e. red betel. Red betel is the type of betel often used as
ornamental plant in the 1990s, but now it has shifted to medicinal function since its introduction
by Sudewo (2010), a medicinal plants producer in Blunyahrejo.
Antibacterial capacity of red betel leaves has proven effective in combating pathogenic
bacteria such as Staphylococcus aureus and Escherichia coli (Juliantina et al., 2009) Ethanolic
extract of red betel leaves also shows antibacterial activities against Pseudomonas aeruginosa
and Shigella dysenteriae (Noor, 2011)
This research is conducted to test the antibacterial activity of the red betel leave extract
against the growth of MRSA. Red betel leave extract is used with methanolic dilution in this
research. Methanol is a polar dilution so it can attract all polar active metabolic substances such
as tannin and flavonoid, thus increasing the antibacterial capacity of the extract (Pradipta,
2007; Wresdiyati (2003).

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Material and Methods

This reaserach is a laboratory experiment using Completely Randomized Design pattern
with 4 repetitions. The design of this research consists of 5 treatment groups namely extract
concentration of 150 mg/mL, 300 mg/mL, 450 mg/mL, 600 mg/mL and negative control (sterile
water).
Preparation of Red Betel Leaves
The red betel leaves were obtained from Simpang Balik village, Wih Pesam sub-district,
Bener Meriah. Geographically, Bener Meriah is located in a tropical region with average
humidity of 75%, at the elevation of 100-1200 m above sea level. The average annual
precipitation ranges from 1000 to 2000 mm. The soil texture and drainage system are good and
the average temperature ranges from 260C to 270C. The leaves used were 500 gr fresh red
betel leaves. The leaves were picked in the morning to allow a proper drying process. Using
clean cutting tools, the picking started from the third leaf below the buds to the ninth leaf in
every sprig. This method was applied due to the leaves’ high content of active substance with
strong scent and bitter flavor( Sudewo, 2010).
Red Betel Leaf Herbarium Test
Herbarium test was performed to identify and ensure the family and species of red betel
plant to be examined based on its morphological characteristics. The result showed that the red
betel used belonged to Piperaceae family and of Piper crocatum species.
The Formulation of Red Betel Leave Methanolic extract
500 gram of fresh red betel leaves was soaked in water for 30 minutes and then washed,
clean, and drained. The leaves were then airdried for 7×24 hours in room temperature with no
direct sunlight, after which 173 gram of dried red betel leaves was obtained. The dried leaves
were ground to a powdery consistency. The powder was soaked in 2 litres of 96% methanol
dilution for 3×24 hours, changing the dilution every 24 hours, and then strained using filter
paper. The filtrate resulted from the straining was vaporized using rotary vacum evaporator at
400C to get a concentrated extract of red betel leaves (Safithri and Fahma, 2005). This extract
was then divided into several testing concentrations.
Phytochemical Screening
Flavonoid Testing. 0.05 gram of red betel extract and 96% methanol were heated for 5
minutes. H2SO4 was then added to the filtrate. Flavonoid compound was indicated by the red
color caused by the addition of H2SO4. Alkaloid Testing. 5 ml chloroform and ammoniac was
added to 0.05 gram of red betel extract.The chloroform fraction was then separated and
acidified using 1 drop of H2SO4 2M. The acid fraction was divided into three cylinders, each of
which was added Dragendorf, Meyer and Wagner reagent. The presence of alkaloid was
indicated by the forming of white sediment in the Meyer reagent, red sediment in Dragendorf
reagent, and brown sediment in Wagner reagent. Tannin Testing. 0.05 gram of red betel extract
was added 12.5 mL of sterile water, and then brought to a boil for 5 menit. The solution was
filtered and 5 drops of FeCl3 1% (b/v) was added to the filtrate. The formed color of dark blue or
greenish black showed the presence of tannin. Steroid and Triterpenoid Testing. 0.05 gram of
red betel extract was added 12.5 mL of ethanol 30% and heated for 5 minutes and strained. Its
filtrate was vaporized and ether was added. Lieberman Buchard reagent (3 drops of acetate
anhydride and 1drop of concentrated H2SO4). The formed red or purple color indicated the
presence of triterpenoid and green showed steroid. Saponin Testing. 0.05 gram of red betel
extract was added 5 mL of sterile water and heated for 5 minutes. It was then shaken for 5
minutes. Saponin test showed a positive result if foam of at least 1 cm high was formed and
stayed stable after being set aside for 10 minutes.
Reidentification of Tested Bacterium
The MRSA isolate was obtained from the stock culture isolated from the pus of diabetic
ulcer patients at the Zainal Abidin Hospital’s Microbiology Laboratory. Next, reidentification of
the tested bacterium was performed through macroscopic and microscopic (Gram Staining)
examination, catalase test, coagulation test, as well as test of resistance to Methicillin (Brown,
2005).
Macroscopic Examination. The bacterium grown in the inoculation medium was
macroscopically identified. The examination comprised shape, surface, border and color of the

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colony growing in the Nutrient Agar medium. Microscopic Examination. This examination
was performed through Gram staining.
Catalase Test. Bacterium culture was smeared on the glass object evenly and then given 1
or 2 drops hydrogen peroxide solution (H2O2) 3%. The catalase activities on the microbes can
be identified by examining whether or not the gas bubbles were formed. Coagulation Test.
Bacterium culture was obtained using sterilized ose and then inoculated into a cylinder
containing 0.5 ml of human plasma. Positive coagulation test was indicated by the forming of
coagulated plasma in the cylinder afte being incuated for 24 hours at 370C. Test of Resistance
to Methicillin. Inocula was gradually suspended into sterile NaCl 0,9% on the wall of the test
tube to produce a fine bacterial suspension. The bacterium was inoculated to a MHA medium by
dipping a sterilized cotton bud into the inocula. The cotton bud was applied onto the
surrounding of the agar surface. The inocula was left to dry for a few minutes at room
temperature. Resistance test was performed by using oxacillin 5 µg antibiotic disc, which was
incubated at 350C. After that, the diameter of the formed inhibition zone was measured using a
vernier caliper and the result was stated in milimeter (mm). Should the inhibition be ≤ 10 mm,
it is indicated that the S. aureus strain is resistant to methicillin/oxacillin.
Antibacterial Activity Test of Red Betel Leaf Methanolic extract against MRSA
This test applied the Kirby-Bauer disc diffusion method using Mueller Hinton Agar (MHA)
medium, in accordance with the procedures of European Committee on Antimicrobial
Susceptibility Testing/EUCAST (2009). Inocula was suspended gradually into sterile NaCl 0.9%
on the wall of the test tube to produce a fine bacterial suspension. Next, bacterial turbidity was
measured using spectrophotometer at the wavelength of 625 nm and absorbency of 0.08-0.1.
The bacterium was inoculated to a MHA medium by dipping a sterilized cotton bud into the
inocula. This testing of red betel leaf methanolic extract used an empty disc of 6 mm in
diameter. The empty disc was soaked in the extract solution of different concentrations for 30
minutes. It was then set on an inoculated petri dish using sterilized tweezers. The negative
control used an empty disc soaked in sterile water. Each treatment was repeated 4 times. The
next step was incubation for 24 hours at 370C.

Results and Discussion

Identification and Extraction of Red Betel Leaves
The identification result of red betel leaves based on the herbarium test performed at the
Herbarium Laboratory of Biologi Department, Math and Science Faculty, Syiah Kuala University,
showed that the fresh red betel leaves collected from Wih Pesam sub-district, Bener Meriah,
were from red betel plant (Piper crocatum). 500 gram of fresh leaves was airdried to produce
173 gram of dried red betel leaves. After being macerated in 2 litres of 96% methanolic dilution
for 3×24 hours, it produced 26.1 gram red betel leaf methanolic extract of blackish green color
and thick consistency. The extract was then divided into concentrations of 150 mg/mL, 300
mg/mL, 450 mg/mL, 600 mg/mL.
Phytochemical Screening
The result of the red betel leaf methanolic extracts phytochemical screening showed the
presence of alkaloid, flavonoid, saponin, triterpenoid, and tannin compounds (Table 1).

Table 1. Result of phytochemical screening of red betel leaf methanolic extracts

No Compound Result*
1 Alkaloid Mayer Reagent -
Dragendorf Reagent +
Wagner Reagent -
2 Flavonoid +
3 Saponin +
4 Triterpenoid +
5 Steroid -
6 Tannin +

*(+) = containing tested compound; (-) = not containing tested compound.

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& The 8th IMT-GT Uninet Biosciences Conference
Banda Aceh, 22-24 November 2012

Flavonoid contained in red betel leaves comprises flavonol, flavanon, isoflavon, auron,
cathechin, antocyanidines dan chalcones (Arishandy, 2010; Lenny, 2006). The total flavonoid
concentrations of the extract of fresh red betel leaf boiling water, the extract of commercial red
betel leaf boiling water, and the methanolic extract of fresh red betel leaves are 0.57% b/b,
0.54% b/b dan 1.89% b/b (Wirdani, 2008). Therefore, the methanolic dilution produced higher
flavonoid compared to the extract of red betel leaf boiled water which constitutes the most
common use of the leaves by Indonesian communities. Flavonoid content was also found in the
red betel leaf ethanolic extract. This is possible because flavonoid is a polar compound and only
dilutes in polar dilutions such as ethanol/methanol (Hasbi 2011). A number of other active
substances such as tannin, saponin and alkaloid were also found in the extract used in the
research, but not in the red betel leaf ethanolic extracts (Hasbi, 2011). The difference can be
caused by a lot of factors such as organ differences, cells, age of plants, seasons, and
geographical location of the plants (Bohm, 2009; Shahidi and Ho, 2000). In addition, the
difference was also the result of the characteristics of a number of active substances against
temperatures or a very low content, thus unable to be detected in the qualitative phytochemical
screening.
Reidentification Result of Methicillin-Resistant Staphylococcus aureus
The result of macroscopic observation on the Nutrient Agar (NA) medium showed that S.
aureus formed a colony of 0.5 mm in size, of white color, round shape with fine border and even
surface. Gram Staining result showed a bacterium in coccus shape, purple color, and forming a
small group resembling grapes which indicated Gram positive bacterium. The coagulation test
using plasma in tubes produces a positive result namely the forming of plasma coagulation.
From the catalase test, a positive result was obtained which was indicated by the forming of gas
bubbles. The result of resistance test of S. aureus to methicillin showed that S. aureus isolates
are resistant to antibiotic methicillin. All the above mentioned results show that the tested
bacterium was MRSA.
Result of Actibacterial Activity Test of Methanolic Extract of Red Betel Leaf Against
MRSA
The antibacterial activity test of red betel leaf methanolic extract against the growth of
MRSA at the 15%, 30%, 45% and 60% concentrations resulted in inhibition zones of average
diameter of 9.0 mm, 11.2 mm, 13.6 mm and 15.7 mm each. The average diameter of the
inhibition zone formed in sterile water was 0 mm. The data obtained was then analyzed using
Analisis of Variance (ANOVA) to examine the impact of each treatment. The result of ANOVA
revealed that there was a significant difference in the antibacterial activity indicated by each
concentration of red betel leaf methanolic extracts. Statistical test was then continued with
Duncan Test at p<0,05.

Table 2. Average diameter of inhibition zone ± deviation standard of red betel leaf methanolic extract
against the growth of MRSA. The average value of inhibition zone was followed by different superscripts
showing significant differences (P<0,05)

Treatment Diameter of inhibition zone in each Average diameter of

repetition (mm) inhibition zone (mm) ±
SD
I II III IV
Negative control 0 0 0 0 0 ± 0,00a
150 mg/ml extract concentration 10 9 8 9 9,0 ± 0,81b
300 mg/ml extract concentration 12 12 11 10 11,2 ± 0,95c
450 mg/ml extract concentration 15 14 13 12,5 13,6 ± 1,10d
600 mg/ml extract concentration 17 16 15 15 15,7 ± 0,95e

.The red betel leaf methanolic extracts of 450 g/ml and 600 mg/ml concentrations could
still be used as antibacterial material against MRSA. This is based on the general standard
stipulated by National Committe for Clinical Laboratory Standards/NCCLS (2002) in Tambekar
and Dahikar (2010), that a bacterium is considered susceptible to antibacterial materials from
plants when the inhibition zone diameter is more than 12 mm. The red betel leaf methanolic
extract of 15% and 30% concentrations could not be used as antibacterial material against
MRSA as its inhibition zone diameter is less than 12 mm.
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The capability of red betel leaf methanolic extracts to combat the growth of MRSA owes to
the active compounds contained in the plant such as flavonoid, alkaloid, tannin, saponin and
triterpenoid. This is in line with the statement of Tsuchiya, Sato and Miyazaki (1996) that 2’, 4’
or 2’, 6’-dihydroxylation of B ring and 5,7- dihydroxylation of A ring within the flavanon
structure is important to anti-MRSA activity. Alcaraz et al., (2000) also reports the importance of
hydroxyl group at 5 position from flavon and in the activity against MRSA, which supports the
finding of Tsuchiya, Sato and Miyazaki (1996).
A research by Sakagami, Mimura and Kajimura (1998) asserts that sophoraflavanon G has
an intensive antibacterial power against MRSA (Tsuchiya dan Linuma, 2000). The effect of
sophoraflavanon G on the fluidity of membranes was studied using liposomal model of
membranes. At the concentration compatible with minimum inhibitory concentration,
sophoraflavanon G was proven to significantly increase the fluorescence polarization of liposome.
The increase showed that sophoraflavanon G reduced the fluidity of the outer and inner layers of
bacterial cell membranes.
According to Aniszewski’s research (2007, alkaloid has antibacterial activities despite of its
unclear mechanisms. However, it is connected to the chemical elements of carbon rings,
aromatic substitution and oxidation nature of alkaloid which can be antibacterial. Alkaloid can
cause bacterial cells lysis and contains toxin which can inhibit bacterial growth.
Tannin is a chelating agent which can cause the cell membrans to shrink hence distorting
the cell’s permeability. Lim et al., (2006) expose that hydrolyzed tannin can slow down the
growth of cells by restricting the synthesis of cell membranes development of the cell’s wall.
Abnormality in the cell membranes then causes a change in the cell’s permeability and death of
the bacteria. Besides through the reaction to the cell’s membranes, the effect of tannin
antimicrobes can also be observed from the enzyme inactivity and destruction or the inactivity
of genetic material function. Tannin also has antibacterial activities related to its ability to set
adhesin and enzyme inactive and join the cell’s wall. Tannin can cause the membranes to burst
(Cowan, 1999).
Saponin can function as antimicrobes. Saponin has molecules that attract water or
hidrophillic and molecules that can dilute lipid or lipophillic, thus decreasing the pressure of cell
surface which eventually causes the destruction of the bacterium. Triterpenoid can actively fight
against bacteria, but its antibacterial mechanism is still really unclear. The antibacterial activity
of triterpenoid is assumed to involve bursting of membranes by the lipophillic componenets
(Cowan, 1999). Triterpenoid compounds can also result in the decreased semipermeability of
cell’s membranes causing the nutrients and enzymes to abandon cytoplasm. Bacterial
metabolism will be hindered resulting in the reduced production of ATP. This causes inhibition of
bacterial growth and reproduction.

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